Nutritional product for a person having ulcerative colitis

ABSTRACT

An enteral nutritional product for a person having ulcerative colitis contains in combination (a) an oil blend which contains eicosapentaenoic acid (20:5n3) and/or docosahexaenoic acid (22:6n3), and (b) a source of indigestible carbohydrate which is metabolized to short chain fatty acids by microorganisms present in the human colon. Preferably the nutritional product also contains one or more nutrients which act as antioxidants.

This application is a divisional of application Ser. No. 09/083,736,filed on May 22, 1998, now U.S. Pat. No. 5,952,314, which is acontinuation-in-part of application Ser. No. 08/221,349, filed on Apr.1, 1994, now U.S. Pat. No. 5,780,451.

TABLE 1 CURRENT DRUG THERAPIES FOR ULCERATIVE COLITIS “non-specifictherapies” DRUG ADMINISTRATION SIDE EFFECTS Anti-inflammatory agentsSalicylates oral, rectal (enemas) secretory diarrhea, 5-ASA (ROWASA)nausea, headache, Sulfasalazine anemia, leakopenia Corticosteroids oral,topical, acne, weight gain, intravenous peptic ulcer, diabetes, glaucomacataracts, osteoporosis, psychosis Immunosuppressive agents Azathioprine(AZA) oral, intravenous bone marrow 6-mercaptopurine suppression,Metronidazole infections, Cyclosporine pancreatitis Prednisone

The present invention relates to a nutritional product for a personhaving ulcerative colitis.

The term “Inflammatory Bowel Disease” is a designation commonly used fortwo related, but distinct, chronic inflammatory conditions affecting thegastrointestinal tract, namely Crohn's disease and ulcerative colitis.Crohn's disease may involve any segment of the gastrointestinal tract,although characteristically the region of greatest involvement is thedistal one quarter of the small intestine and the proximal colon. Inulcerative colitis the inflammation is, by definition, limited to themucosa of the large bowel. However, the present invention is concernedonly with nutritional support for a person having ulcerative colitis.The primary cause of ulcerative colitis is not currently known.

At the present time, there is no medical cure for ulcerative colitis andthis chronic condition may lead to total proctocolectomy. Currentmedical treatment is directed toward decreasing the number, frequencyand severity of acute exacerbations of inflammatory bowel disease andpreventing secondary complications, but at best, the results aredisappointing. Long term use of corticosteroids to downregulate theinflammatory response is a common approach to the control of intestinalinflammation. Steroids are considered to exert their antiinflammatoryeffects through inhibition of the release of free arachidonic acid frommembrane phospholipids; Historically the long term use ofimmunosuppressive agents (steroids) is associated with chronic sideeffects such as those presented in Table 1.

Sulfasalazine is widely used to treat victims of ulcerative colitis.Sulfasalazine's pharmacologic effects include alterations in thebacterial flora of the gut, increased colonic absorption of fluids andelectrolytes, decreases in the number of B cells, interference withlymphocyte activation and natural killer activity, and inhibition ofantibody secretion. The overall usefulness of sulfasalazine has beensomewhat undermined by a high degree of intolerance and a frequentoccurrence of adverse reactions in the patient population such as thosepresented in Table 1.

Antibiotics are used intermittently, particularly in the presence ofsevere exacerbations as are other drugs including antispasmodics andanticholinergics. It has been reported by Rosenberg et al., “NutritionalAspects of Inflammatory Bowel Disease”, ANNUAL REVIEW OF NUTRITION, Vol.5, pages 463-484, at 467 (1985) that many drug therapies used ininflammatory bowel diseases may have negative effects on nutritionalstatus. For example, high daily doses of corticosteroids can exert anadditional catabolic effect on patients who may already be under stress,and may inhibit calcium absorption by the intestine. Another example ofa potentially negative drug-nutrient interaction is the interferencewith folate absorption by sulfasalazine via a mechanism of competitiveinhibition.

Therapy for severe attacks of ulcerative colitis frequently includesspecial nutritional support, especially when surgical intervention isplanned. Total parenteral nutrition was initially used to improvenutritional status, but later was used to enhance “bowel rest” andinduce clinical remission to avoid total proctocolectomy. However;Gonzalez-Huix et al., “Enteral versus Parenteral Nutrition as AdjunctTherapy in Acute Ulcerative Colitis”, THE AMERICAN JOURNAL OFGASTROENTEROLOGY, Vol. 8, No. 2, pages 227-232 (1993) reports theresults of a study which suggests that total enteral nutrition is safeand nutritionally effective in severe attacks of ulcerative colitis.This publication suggests total enteral nutrition should be regarded asthe most suitable type of nutritional support in these patients. Theenteral nutritional product used in this published study was Edanec HNfrom UNIASA, Granada, Spain which was described in the publication asset forth below in Table 2.

TABLE 2 EDANEC HN NUTRIENT AMOUNT PER 1000 ml Nitrogen (g) 8.73 Lipids(g) 36.20 Carbohydrates (g) 110.20 Energy (Kcal) 984.85 E/N ratio(nonprotein kcal/g N) 87.81 Energy source Nitrogen Intact milk proteinFat Long Chain Triglycerides Carbohydrate Maltodextrins Na (mmol) 36.00K (mmol) 32.00 Calcium (mmol) 3.00 Magnesium (mmol) 3.00 Phosphate(mmol) 12.00 Vitamins Upper limit of RDA Trace-elements Upper limit ofRDA

Gonzalez-Huix et al., compared the effects of total enteral nutritionand total parenteral nutrition in patients with acute ulcerativecolitis. The final conclusions of their trials were that totalparenteral nutrition does not have a primary therapeutic effect on theinflammatory process, and that “bowel rest” is not essential for themanagement of acute ulcerative colitis. The main reluctance to useenteral feeding in severe ulcerative colitis has been the possibility ofworsening diarrhea. Gonzalez-Huix et al. reported that only one patientout of 23 fed enterally developed diet-related diarrhea. Although aregular diet may be well-tolerated in ulcerative colitis, patients tendto reduce food intake unless they are persistently encouraged to eat. Inthese circumstances, tube feeding has been used to guarantee adequateenergy and nutrient supply.

The UNIASA product, Edanec HN, differs considerably from the nutritionalproduct of the present invention. For example, the new product of thepresent invention has a caloric density of 1.29 kcal/ml while Edanec HNhas a caloric density of 0.98 kcal/ml. Our product also is lower in fat,containing approximately 21.9 g Fat/1000 kcal while Edanec HN containsapproximately 36.7 g Fat/1000 kcal. The nutritional product of thepresent invention also contains fish oil as a source of eicosapentaenoicacid (20:5n3) and docosahexaenoic acid (22:6n3) as well as dietaryfibers such as gum arabic and indigestible oligosaccharides such asfructooligosaccharides (FOS) and xylooligosaccharides (XOS). Theseingredients are crucial for a product developed for a patient withulcerative colitis.

Ulcerative colitis afflicts persons as young as 5 years old. Onset ofsymptoms of inflammatory bowel disease occurs before age 20 in about 40%of patients. The biggest problem in the management of ulcerative colitisin young persons is almost invariably poor dietary compliance. It hasbeen observed by Sutton, “Nutritional Needs of Children withInflammatory Bowel Disease”, NUTRITION, Vol. 18, No. 10, pages 21-25(1992) that deficiencies of micronutrients are individually determinedand relate to disease activity and site as well as dietary intake.Sutton recommends a multivitamin/mineral tablet which meets 100-150% ofthe Recommended Daily Allowance. This publication further reports that:(a) deficiencies of water-soluble nutrients such as folate, B₁₂, biotin,vitamin C, niacin, riboflavin, and B₆ have been reported in patients whoeliminated foods such as milk, fruits and vegetables due to intolerance;(b) deficiencies of fat-soluble nutrients such as vitamins A, E and Khave been reported in patients having fat malabsorption due to severeileac disease or resection; and (c) deficiencies of minerals and traceminerals such as calcium, iron, zinc, copper and chromium, result frominadequate intake and/or reduced absorption.

Similar nutritional deficiencies in inflammatory bowel disease patientshave been reported by Rosenberg et al., “Nutritional Aspects ofInflammatory Bowel Disease”, ANNUAL REVIEW OF NUTRITION, Vol. 5, pages463-484 (1985). Rosenberg, et al. describe the problems of proteincalorie malnutrition and deficiency of micronutrients ongastrointestinal function and structure in these terms: The patient withinflammatory bowel disease who becomes significantly malnourished mayenter a vicious cycle where secondary effects of malnutrition orgastrointestinal function and structure may lead to a further increasein gastrointestinal symptoms and malabsorption, which further worsensnutrient balance. In addition, it may be assumed that malnutrition willsignificantly depress the patient's ability to heal the inflammation andstructural changes in the bowel. The overall therapeutic strategy mustbe to ensure adequate intake of nutrients while modifying dietary intaketo decrease gastrointestinal symptoms.

The impact of ulcerative colitis on nutritional status can be highlysignificant, particularly in the pediatric age group, in whom proteinand calorie requirements for growth are not likely to be met by ordinarydietary means. There is increasing evidence that a good therapeuticresponse can be achieved in ulcerative colitis by dietary treatmentalone. Many dietary regimens have fallen short of expectations and havenot been uniformly effective in promoting weight gain and wound healingor in maintaining optimal nutritional status in patients with ulcerativecolitis.

The features of the present invention which are believed to be novel areset forth with particularity in the appended claims. The presentinvention may be understood by referring to the following detaileddescription, taken in accordance with the accompanying drawing FIGS.1-5, which are all charts presenting the results of experiments relatingto the present invention.

The major advantages of using a specially formulated enteral diet toinduce remission of active disease include the virtual absence of sideeffects, possible decreased dosage of prescribed drugs and improvednutritional status of adults and children. In order to understand andevaluate the effects of polymeric diet(s), various nutrients such as n-3fatty acids, nutrients which function as antioxidants, and short chainfatty acids (SCFAs) must be assessed as to their ability to decreasedisease activity in ulcerative colitis and allow for mucosal repair.

Increasing interest has been generated in the use of enemas/irrigationsolutions containing buffered, physiologic levels of SCFAs for thetreatment of diversion colitis and ulcerative colitis. Diversion colitisis an inflammatory process arising in segments of the colorectum atvarious intervals after surgical diversion of the fecal stream. Theendoscopic appearance is similar to those of active Crohn's Disease andulcerative colitis. Glotzer et al., “Proctitis and Colitis FollowingDiversion of the Fecal Stream”, GASTROENTEROLOGY Vol. 80, pages 438-441(1981). The cause of this condition is not known, but one mechanism hasbeen postulated; a nutritional deficiency of the colonic epithelium,specifically due to the absence of SCFAs normally present in coloniccontents, Komorowski, “Histologic Spectrum of Diversion Colitis”AMERICAN JOURNAL OF SURGICAL PATHOLOGY, Vol. 14, page 548 (1990),Roediger, “The Starved Colon—Diminished Mucosal Nutrition, DiminishedAbsorption, and Colitis”, DISEASES OF THE COLON AND RECTUM, Vol. 33,pages 858-862 (1990). Harig et al., “Treatment of Diversion Colitis withShort-Chain-Fatty Acid Irrigation”, NEW ENGLAND JOURNAL OF MEDICINE,Vol. 310, pages 23-28 (1989) tested this hypothesis by assessing whetherirrigation with SCFAs could ameliorate inflammation in four patientswith diversion colitis. These patients were administered SCFAs twicedaily for 2-3 weeks with 60 mL of an enema solution comprising aphysiologic mixture of SCFAs as sodium salts. After 2-3 weeks oftherapy, macroscopic and histological resolution of inflammation wasevident. An impaired utilization of SCFAs has also been implicated inulcerative colitis which suggests that diminished intracellular energyproduction may be important in the inflammatory process, Roediger, “TheColonic Epithelium in Ulcerative Colitis: an Energy DeficiencyDisease?”, THE LANCET, Vol. 2, pages 712-715 (1980). Vernia et al.,“Fecal Lactate and Ulcerative Colitis”, GASTROENTEROLOGY, Vol. 95, pages1564-1568 (1988); and Vernia et al., “Organic Anions and the Diarrhea ofInflammatory Bowel Disease”, DIGESTIVE DISEASES AND SCIENCES, Vol. 33,pages 1353-1358 (1988) have shown that fecal water from patients withulcerative colitis contains reduced concentrations of SCFAs as well asmarkedly increased lactate and low pH. In a study by Breuer et al.,“Rectal Irrigation with Short-Chain Fatty Acids for Distal UlcerativeColitis” (preliminary report), DIGESTIVE DISEASES AND SCIENCES, Vol. 36,pages 185-187 (1991), relates an investigation of large bowel irrigationwith SCFAs in patients with ulcerative colitis. It was found that 9 outof 10 patients completing the study were judged to be at least muchimproved and showed a significant change in mean disease activity indexscore and mucosal histology score. Recently Senagore et al.,“Short-Chain Fatty Acid Enemas: a Cost Effective Alternative in theTreatment of Nonspecific Proctosigmoiditis”, DISEASES OF THE COLON ANDRECTUM, Vol. 35, page 923 (1992), confirmed the results of Breuer et al.demonstrating an 80 percent response rate in patients with idiopathicproctosigmoiditis. This study indicates that administering a solution ofSCFAs similar to Harig et al. for six weeks was equally efficacious tocorticosteroid or 5-ASA enemas for the treatment of proctosigmoiditis ata significant cost savings. Scheppach et al., “Effect of Butyrate Enemason the Colonic Mucosa in Distal Ulcerative Colitis”, GASTROENTEROLOGY,Vol. 103, pages 51-56 (1992) investigated the use of butyrate enemasalone rather than the SCFA mixture to treat ten patients with distalulcerative colitis in a placebo-controlled, single-blind, randomizedtrial. The authors concluded that markedly improved disease activityindex and histological parameters suggesting that the effect of a SCFAmixture on the inflamed mucosa in ulcerative colitis is largelyattributable to its butyrate moiety.

It is unlikely that short chain fatty acids added directly to an enteralproduct would reach the large bowel. Also, the stability of thesecompounds in a nutritional product is questionable. However, thenutritional product of the present invention takes advantage of thepositive effect of SCFAs by providing dietary fiber or indigestibleoligosaccharides.

For the purpose of the patent the following terms are defined asfollows:

Dietary Fiber—A material that contains a large carbohydrate moiety(Degree of polymerization greater than 20 and/or a molecular weightgreater than 3,600) that is resistant to endogenous digestion in thehuman upper digestive tract.

Indigestible Oligosaccharide—A small carbohydrate moiety (Degree ofpolymerization less than 20 and/or a molecular weight less than 3,600)that is resistant to endogenous digestion in the human upper digestivetract.

Indigestable Carbohydrate—A term used to encompass both dietary fiberand indigestible oligosaccharides.

Certain of the organisms that inhabit the large bowel can utilizedietary fiber (eg, pectin and gum arabic) and indigestibleoligosaccharides (eg, fructooligosaccharides and xylooligosaccharides)as an energy source. Smith et al., “Introduction to Metabolic Activitiesof Intestinal Bacteria”, AMERICAN JOURNAL OF CLINICAL NUTRITION, Vol.32, pages 149-157 (1979); Miller et al., “Fermentation by SaccharolyticIntestinal Bacteria”, AMERICAN JOURNAL OF CLINICAL NUTRITION, Vol. 32,pages 164-172 (1979); Cummings., “Fermentation in the Human LargeIntestine: Evidence and Implications for Health”, THE LANCET, Vol. 1,pages 1206-1209 (1983); Titgemeyer et al., “Fermentability of VariousFiber Sources by Human Fecal Bacteria In Vitro”, AMERICAN JOURNAL OFCLINICAL NUTRITION, Vol. 53, pages 1418-1424 (1991). The microorganismsderive energy from the carbohydrate sources through a process referredto as anaerobic fermentation. During fermentation, the microorganismsproduce SCFAs (eg, acetate, propionate, butyrate) as the major endproducts. Salyers et al., “Fermentation of Mucin and PlantPolysaccharides by Strains of Bacteroides from the Human Colon”, APPLIEDAND ENVIRONMENTAL MICROBIOLOGY, Vol. 33, pages 319-322 (1977); Mitsuokaet al., “Effect of Fructo-oligosaccharides on Intestinal Microflora”,DIE NAHRUNG, Vol. 31, pages 427-436 (1987); Tokunaga et al., “Influenceof Chronic Intake of a New Sweetener Fructooligosaccharide (Neosugar) ongrowth and Gastrointestinal Function of the Rat”, JOURNAL OF NUTRITIONALSCIENCE AND VITAMINOLOGY, Vol. 32, pages 111-121 (1986).

As an indirect source of SCFAs, dietary fiber and indigestibleoligosaccharides (indigestable carbohydrate) can elicit certainmetabolic benefits. Total parenteral nutrition (TPN) or theadministration of a fiber free liquid diet leads to reduced colonic cellproliferation and atrophy. Janne et al., “Colonic Mucosal AtrophyInduced by a Liquid Elemental Diet in Rats”, DIGESTIVE DISEASES, Vol.22, pages 808-812 (1977); Morin et al., “Small Intestinal and ColonicChanges Induced by a Chemically Defined Diet”, DIGESTIVE DISEASESCIENCE, Vol 25, pages 123-128 (1980); Sircar et al., “Effect ofSynthetic Diets on Gastrointestinal Mucosal DNA Synthesis in Rats”,AMERICAN JOURNAL OF PHYSIOLOGY, Vol. 244, pages G327-G335 (1983); Ryanet al., “Effects of Various Diets on Colonic Growth in Rats”,GASTROENTEROLOGY, Vol. 77, pages 658-663 (1979); Storme et al., “TheEffects of a Liquid Elemental Diet on Cell Proliferation in the Colon ofrats”, CELL TISSUE RESEARCH, Vol. 216, Pages 221-225 (1981). Suchatrophy could be prevented with the use of indigestible carbohydrate.Indigestible carbohydrate, through the production of SCFAs during theirfermentation, can stimulate colonic epithelial cell proliferation.Goodlad et al., “Proliferation Effects of Fibre on the IntestinalEpithelium”, GUT, Vol. 28 pages 221-226 (1987); Kripe et al.,“Stimulation of Intestinal Mucosal Growth with Intracolonic Infusion ofShort-Chain fatty Acids”, JOURNAL OF PARENTERAL AND ENTERAL NUTRITION,Vol. 13, pages 109-116 (1989); Scheppach et al., “Effect of Short-chainFatty Acids on the Human Colonic Mucosa In Vitro”, JOURNAL OF PARENTERALAND ENTERAL NUTRITION, Vol. 16, pages 43-48 (1992); Sakata.,“Stimulatory Effect of Short-chain Fatty Acids on Epithelial CellProliferation in the Rat Intestine: A Possible Explanation for TrophicEffects of Fermentable Fibre, Gut Microbes and Luminal Trophic Factors”,BRITISH JOURNAL OF NUTRITION, Vol. 58, pages 95-103 (1987); Thomas etal., “Effect of enteral Feeding on Intestinal Epithelial Proliferationand fecal Bile Acid Profiles in the Rat”, JOURNAL OF PARENTERAL ANDENTERAL NUTRITION, Vol. 17, pages 210-213 (1993). A recent animal studyalso has demonstrated the benefit of an indigestible carbohydrate in thetreatment of experimental colitis. Rolandelli et al., “Comparison ofParenteral Nutrition and Enteral Feeding with Pectin in ExperimentalColitis in the Rat”, AMERICAN JOURNAL OF CLINICAL NUTRITION, Vol. 47,pages 15-21 (1988). Specifically, the degree of bowel injury inexperimental colitis was decreased when rats were fed an enteral dietsupplemented with pectin, which is a dietary fiber. Improvements inoutcome may have been due to the SCFAs produced during the fermentationof pectin.

EXPERIMENT 1

In the first experiment the objective was to determine short chain fattyacid production from a variety of indigestible oligosaccharides duringfermentation with mixed human fecal microbiota. Several indigestibleoligosaccharides were tested including FOS, Raftilose® and XOS. FOS is afructooligosaccharide produced on a commercial scale by fermentinggranulated sucrose in water with a pure strain of Aspergillus niger. Theorganism produces a fructosyltransferase enzyme which links additionalfructose units onto the fructose end of sucrose molecules to produce1-kestose (GF₂), nystose (GF₃) and 1^(F)-β-fructo-furanosylnystose(GF₄). Raftilose® is a fructooligosaccharide produced via enzymatichydrolysis of inulin, which is marketed by Rhone-Poulenc (RaffinerieTirlemontoise SA). The hydrolysis results in a wide array ofoligosaccharides such as GF₂, GF₃ and GF₄ as well as oligosaccharidescontaining just fructose (F₃, F₄, F₅, etc.). XOS is axylooligosaccharide produced via enzymatic hydrolysis of xylan. Theprimary ingredients of XOS are xylobiose, xylotriose and xylotetrose.

The fermentation medium used in this first experiment is described inTable 3, and the anaerobic dilution solution used in this experiment isdescribed in Table 4.

TABLE 3 IN VITRO FERMENTATION MEDIUM COMPOSITION^(a) INGREDIENT AMOUNT(%) Substrate (w/v) 1.0 Salts A^(b) (v/v) 33.0 Salts B^(c)(v/v) 33.0Salts SL6^(d) (v/v) 1.0 Vitamin mix^(e) (v/v) 2.0 Hemin Solution^(f)(v/v) 0.25 Resazurin solution^(g) 0.10 (v/v) Yeast extract (w/v) 0.05Trypticase (w/v) 0.05 Na₂CO₃ (w/v) 0.40 cysteine HCl H₂O (w/v) 0.05 SCFAmix^(h) (v/v) 0.04 d H₂O (v/v) 31.88 ^(a)Media will be prepared asfollows. All ingredients except substrate, vitamin mix, Na₂CO₃, cysteineHCl and SCFA mix will be dissolved via boiling and then cooled to <50°C. through bubbling with CO₂. Na₂CO₃ and the SCFA mix will then be addedand bubbled with CO₂ until the solution # is 30° C. Substrate will thenbe added and the solution autoclaved for 15 min at 121° C. (15 psi).Cysteine HCl and the vitamin mix will be added to the solution prior todispensing. ^(b)NaCl, 27.0 g; KH₂PO₄, 13.5 g; CaCl₂.H₂O, 0.8 g;MgCl.6H₂O, 0.6 g; MnCl₂.4H₂O, 0.3 g; CoCl₂.6H₂O, 0.3 g; (NH₄)₂SO₄, 27.0g; d H₂O, 0.5 l. ^(c)K₂HPO₄, 13.5 g; d H₂O, 5.0 l. ^(d)EDTA disodiumsalts, 0.25 g; FeSO₄.7H₂O, 0.1 g; d H₂O, 450 ml; Minerals SL6 solution,50 ml (ZnSO₄.7H₂O, 0.04 g; MnCl₂.4H₂O, 0.012; H₃PO₄, 0.12 g; CoCl₂.6H₂O,0.08 g; CuCl₂ 2H₂O, 0.004 g; NiCl₂.6H₂O, 0.008 g; Na₂MoO₄.2H₂O, 0.012 g;d H₂O, 400 ml). ^(e)Thiamine-HCl, 0.05 g; pantothenic acid, 0.05 g;niacin, 0.05 g; pyridoxine, 0.05 g; riboflavin, 0.05 g; folic acid, 1.25mg; biotin 1.25 mg; PABA, 2.5 mg; Vitamin B₁₂, 0.125 mg; d H₂O, 495 ml;Vitamin K₁ solution, 5.0 ml (vitamin K₁, 125 μl; 95% ethanol, 25.0 ml).^(f)hemin, 50 mg; 1N NaOH, 1 ml; d H₂O, 99 ml. ^(g)See Table 4^(h)N-valeric acid, 0.2 ml; isovaleric acid, 0.2 ml; isobutyric acid,0.2 ml; DL-α-methylbutyric acid, 0.2 ml.

TABLE 4 ANAEROBIC DILUTION SOLUTION^(a) (1 Liter) INGREDIENT AMOUNTMineral solution1^(b) 37.5 ml Mineral solution2^(c) 37.5 ml Resazurinsolution (.1% w/v)^(d) 1.0 ml NaHCO₃ 6.37 g d H₂O (sonicated) 924.0 mlcysteine HCl.H₂O 0.5 g ^(a)Mix minerals 1 and 2, resazurin and water,saturate with carbon dioxide, and add NaHCO₃ and autoclave. Add 0.5 g ofcysteine HCl to cooled solution. ^(b)K₂HPO₄, 0.6 g; Na Citrate.2H₂O, 0.2g; d H₂O, 100 ml. ^(c)NaCl, 1.2 g; (NH₄)SO₄, 1.2 g; KH₂PO₄, 0.6 g;CaCl₂, 0.12 g; MgSO₄.7H₂O, 0.25 g; Na Citrate.2H₂O, 2 g; d H₂O, 2 g; dH₂O 100 ml; (dissolve salts in H₂O in above order). ^(d)Resazurin, 0.05g; d H₂O, 50 ml.

The indigestible oligosaccharides were fermented in vitro for 3, 6, 12,and 24 hours with mixed human fecal microbiota. McBurney et al., “Effectof Human Fecal Inoculum on In Vitro Fermentation Variable”, BRITISHJOURNAL OF NUTRITION, Vol.58, pages 233-243, (1987). Fermentations wererepeated with 3 donors because this is the minimal number required tomake accurate extrapolations to the general population. McBurney et al.,“Effect of Human Fecal Donor on In Vitro Fermentation Variables”,SCANDINAVIAN JOURNAL OF GASTROENTEROLOGY, Vol. 24, pages 359-367,(1989). Briefly, a 0.115 g sample (dry weight) was weighed into a balchtube. Approximately 24 hours before the start of the incubation 10 mL ofthe fermentation medium described in Table 3 was added to the samples sothat the samples were hydrated when the inoculum was added. The redoxpotential of the contents of the tubes was reduced, the tubes werecapped with one-way valves and stored overnight in the refrigerator. Oneto two hours before inoculation, the tubes were placed in a 37° C. waterbath.

Fresh human feces was collected from three healthy individuals. Eachfecal sample was collected into a plastic bag. Air was expressed fromthe bag and an aliquot taken and mixed (blended under CO₂) with theanaerobic dilution solution described in Table 4 (40 g feces/360 mLanaerobic dilution solution; 1×10). The solution was filtered through 2layers of cheese cloth and the filtrate served as inoculum. Onemilliliter of this inoculum was injected into each tube. Tubes wereswirled at regular intervals. The fermentation was terminated at theappropriate time point (h) by opening the tubes and adding 2 mL 25%m-phosphoric acid.

Analysis of acetate, propionate and butyrate was conducted according toMerchen et al., “Effect of Intake and Forage Level on Ruminal andTurnover Rates, Bacterial Protein Synthesis and Duodenal Amino AcidFlows in Sheep”, JOURNAL OF ANIMAL SCIENCE, Vol. 62, pages 216-225(1986). Briefly, an aliquot from the balch tube was acidified with 6NHCl and centrifuged at 31,000×g for 20 minutes Concentrations ofacetate, propionate and butyrate were determined in the supernatantusing a Hewlett-Packard 5890A gas chromatograph and a column (180 cm×4mm id) packed with 20% Tween 80—2% H₃PO₄ on 60 to 80 mesh Chromosorb W(Supelco Inc, Bellefonte, Pa., U.S.A.). Nitrogen was used as a carriergas with a flow rate of 70 mL/minutes. Oven temperature was 120° C. anddetector and injector temperatures were 200° C. Lactate was determinedcalorimetrically using a method described in Barker et al., “TheColorimetric Determination of Lactic Acid in Biological Material”,JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 138, page 535, (1941).

The experiment was analyzed as a randomized complete block with fecaldonor serving as the block. Treatments, which were arranged factorially,included substrate and length of fermentation. All analyses wereperformed using the General Linear Models procedure of StatisticalAnalysis Systems (SAS). Least significant difference (LSD) values forseparating treatment means at P<0.05 was 2.83 times the standard errorof the mean (SEM).

SCFA production (acetate, propionate, butyrate and lactate) during invitro fermentation of the oligosaccharides is presented in Table 3. Fourtime points were studied and include 3, 6, 12 and 24 hours. Retentiontime in the large bowel of humans will dictate the length offermentation in vivo. In cases where retention time is great, the extentof substrate fermentability will be a factor which most influences SCFAproduction. If retention time is short, the rate of substratefermentation becomes more important. Since retention times can differsignificantly in an in vivo situation it is necessary to monitorsubstrate degradation over time in vitro in order that comparisons canbe made.

Fermentation of all oligosaccharides was rapid, essentially beingcomplete by 6 hours for the fructooligosaccharides (FOS and Raftilose)and by 12 hours for XOS. The results are presented in Table 5. It isrecommended that the 6 hour and 12 hour values be used to estimate thecomposition of the end-products for the fructooligosaccharides and theXOS, respectively, even though retention times in the large bowel can beconsiderably longer. At later time points it becomes apparent thatlactate is being converted to propionate and acetate to butyrate.Interconversion in a closed in vitro system can be a problem withrapidly fermented substrates. It does not reflect the true state of thelarge bowel where the fatty acids are continually absorbed.

TABLE 5 SHORT CHAIN FATTY ACID PRODUCTION DURING 3, 6, 12 AND 24 H invitro FERMENTATION OF VARIOUS OLIGOSACCHARIDES SUB- SHORT CHAIN FATTYACID^(a) Total STRATE HOUR Acetate Propionate Butyrate Lactate SCFA^(b)FOS 3 1.49 .20 .23 .45 2.37 6 3.61 .54 .87 1.19 6.21 12 3.67 1.01 1.64.54 6.86 24 3.20 1.09 2.09 .01 6.39 Rafti- 3 1.42 .20 .27 .47 2.36lose ® 6 3.49 .53 .92 1.28 6.22 12 3.68 .98 1.70 .59 6.95 24 3.09 1.052.1 .01 6.30 XOS 3 1.21 .15 .13 .14 1.63 6 4.12 .58 .58 .47 5.75 12 5.90.97 1.1 .74 8.72 24 5.53 .96 1.5 .05 8.10 Statistics SEM .13 .08 .08 .08LSD^(c) .37 .23 .23 .23 ^(a)Calculated as (mmol fatty acid in incubationtube minus mmol fatty acid in blank tube) divided by original substratedry matter (DM) and expressed as mmol/g substrate DM. ^(b)Sum ofacetate + propionate + butyrate + lactate and expressed as mmol/gsubstrate DM. ^(c)Differences between mean values within a columngreater than the specified LSD are significantly different P < .05.

As is typically found with in vitro fermentations using human fecalinoculum or in analysis of fecal samples, acetate was the short chainfatty acid found in the highest concentration. Titgemeyer et al.,“Fermentability of Various Fiber Sources by Human Fecal Bacteria InVitro”, AMERICAN JOURNAL OF CLINICAL NUTRITION, Vol. 53, Pages1418-1424, (1991). Baldwin., “Energy Metabolism in Anaerobes”, AMERICANJOURNAL OF CLINICAL NUTRITION, Vol. 23, pages 1508-1513, (1970).determined that acetate, propionate and butyrate account for 83% of theSCFAs produced during anaerobic fermentation by large bowel microflora,and the remaining SCFAs are distributed among isovaleric, isobutyric,valeric, lactic, formic and succinic acids. In this study, aconsiderable amount of lactate was found, particularly duringfermentation with FOS and Raftilose. It has been documented that theoligosaccharides used in this study serve as an energy source forBifidobacteria and that there consumption will lead to the selectivegrowth of this organism in the GI tract. Okazaki et al., “Effects ofXylooligosaccharides on the Growth of Bifidobacteria”, BIFIDOBACTERIAMICROFLORA, Vol. 9, page 77, (1990); Mitsuoka et al., “Effects ofFructo-oligosaccharide on Intestinal Microflora”, DIE NAHRUNG, Vol. 31,pages 427-436, (1987). The primary end products produced byBifidobacteria during fermentation are acetate and lactate. Miller etal., “Fermentations by Saccharolytic Intestinal Bacteria”, AMERICANJOURNAL OF CLINICAL NUTRITION, Vol. 32, pages 164-172, (1979). The factthat these oligosaccharides serve as an energy source for theBifidobacteria could explain the elevated levels of lactate found inthis study.

Total short-chain fatty acid production was greater for thexylooligosaccharides (XOS) compared to the fructooligosaccharides (FOSand Raftilose). The primary factor effecting the quantity of SCFAsproduced during fermentation is the fermentability of the substrate. Itis assumed that the oligosaccharides are completely fermented in thissystem. However, the yield of SCFAs (mol) from a substrate is dependentnot only on the weight of the substrate fermented but also on theaverage molecular weight of the oligosaccharide component sugars. Onecan assume that the fermentation of one monosaccharide molecule canresult in either two acetate, two propionate, two lactate or onemolecule of butyrate. The molecular weight of the components of thefructooligosaccharides (glucose and fructose, 180) is greater than themolecular weight of xylose (150) which is the monomeric component ofXOS. Subsequently, on an equivalent weight basis, there are more molesof monosaccharide molecules with the xylooligosaccharide compared to thefructooligosaccharide. This would explain the greater production of SCFAwith the XOS compared to the fructooligosaccharides. Lastly, thequantity and profile of SCFAs produced was virtually identical betweenthe two fructooligosaccharides (Raftilose and FOS). While thesefructooligosaccharides differ to some extent in their chemicalcomposition, it is apparent that they are metabolized similarly in thisin vitro fermentation system.

In Experiment 1, the in vitro fermentability of three indigestibleoligosaccharides was evaluated. Fermentation of the indigestibleoligosaccharides was rapid and essentially complete by 12 hours. Suchcompounds may serve as an indirect energy source, particularly for thelarge bowel. By serving as an energy source the oligosaccharides may beuseful in preventing large bowel atrophy associated with the feeding ofsemi-elemental and elemental diets. Through the production of SCFAs, theindigestible oligosaccharides also may be useful in the treatment ofinflammatory bowel disease (ulcerative colitis). Also, considering theirability to serve as energy substrates for the anaerobic flora of thelarge bowel, particularly the Bifidobacteria, these compounds may beuseful in promoting the restoration of normal flora following antibiotictherapy or maintaining a normal flora in patients consuming enteraldiets. This may enhance colonization resistance to pathogens such as C.difficile. It is believed to be an important feature of the nutritionalproduct of the present invention that it contains a source ofindigestible carbohydrate which is metabolized to SCFAs bymicroorganisms present in the human colon and which comprise at leastone material selected from the group consisting of dietary fibers andindigestible oligosaccharides.

It is commonplace in western cultures for the predominant sources oflipids in the diet to be vegetable sources, such as corn or sunflowers,which provide relatively high amounts of linoleic acid (18:2n6).Linoleic acid can be metabolized to arachidonic acid (20:4n6) and henceto dienoic eicosanoids, such as prostaglandin E₂ (PGE₂), thromboxane A₂(TxA₂), and leukotriene B₄ (LTB₄). On the other hand, the predominantpolyunsaturated fatty acids present in fish oils are eicosapentaenoicacid (20:5n3) and docosahexaenoic acid (22:6n3). Eicosapentaenoic acid(20:5n3), which is not present in vegetable oils, has been shown to bemetabolized to a family of trienoic eicosanoids, for example,prostaglandin E₃ (PGE₃), thromboxane A₃ (TxA₃) and also leukotriene B₅(LTB₅) which have biological properties that are subtly different fromthose of the arachidonic acid (20:4n6) metabolites.

Although the primary etiology of ulcerative colitis is unknown, growingevidence supports a pathogenetic role of arachidonic acid derivedinflammatory mediators in this disorder. Eicosanoid formation isincreased in specimens from human diseased tissues. Sharon et al., “Roleof Prostaglandins in Ulcerative Colitis. Enhanced production duringActive Disease and Inhibition by Sulfasalazine”, GASTROENTEROLOGY, Vol.75, pages 638-640 (1978); Ligumsky et al., “Enhanced Thromboxane A₂ andProstacylcin Production by Cultured Rectal Mucosa in Ulcerative Colitisand its Inhibition by Steroids and Sulfasalazine”, GASTROENTEROLOGY,Vol. 81, pages 444-449 (1981); Sharon et al., “Enhanced Synthesis ofLeukotrine B₄ by Colonic Mucosa in Inflammatory Bowel Disease”,GASTROENTEROLOGY, Vol. 86, pages 453-460 (1984). Luminal eicosanoidrelease measured in vivo in patients with active ulcerative rectocolitisis enhanced. Lauristen et al., “In Vivo Effects of Orally AdministeredPrednisolone on Prostaglandin and Leukotriene Production in UlcerativeColitis”, GUT, Vol. 28 pages 1095-1099 (1987); Lauritsen et al., “InVivo Profiles of Eicosanoids in Ulcerative Colitis, Crohn's Colitis andClostridium Difficile Colitis”, GASTROENTEROLOGY, Vol. 95, pages 11-17(1988). Furthermore, animal and clinical work from a number oflaboratories suggests that elevated levels of leukotriene B₄,thromboxane and platelet activating factor participate in thedevelopment of chronic lesions. Seidman, “Nutritional Management ofInflammatory Bowel Disease”, GASTROENTEROLOGY CLINICS OF NORTH AMERICA,Vol. 17, pages 129-155 (1989); Dudrick et al., “Nutritional Managementof Inflammatory Bowel Disease”, SURGICAL CLINICS OF NORTH AMERICA, Vol.71, No. 3, pages 609-623 (1991); Teahon et al., “The Role of Enteral andParenteral Nutrition in Crohn's Disease and Ulcerative Colitis”,PROGRESS IN INFLAMMATION BOWEL DISEASE, Vol. 12, No. 2, pages 1-4(1991); Vilaseca et al., “Participation of Thromboxane and OtherEicosanoid Synthesis in the Course of Experimental InflammatoryColitis”, GASTROENTEROLOGY, Vol. 98, pages 269-277 (1990).

Local eicosanoid generation by the gastrointestinal mucosa is modulatedby intraluminal, neural and hormonal factors. Among the intraluminalfactors, the diet might have a significant relevance in the regulationof mucosal eicosanoid biosynthesis, since the dietary intake ofprecursor fatty acids could directly influence the rate and pattern ofeicosanoid generation. Within the gastrointestinal tract, prostaglandinsderived from arachidonic acid have potent pro-inflammatory actions andcan alter motility, fluid secretion and electrolyte transport. Donowitz,“Arachidonic Acid Metabolites and Their Role in Inflammatory BowelDisease. An Update Requiring Addition of a Pathway”, GASTROENTEROLOGY,Vol. 88, pages 580-587 (1985). In contrast, the lipoxygenase metabolites(leukotrienes) stimulate locomotion, superoxide production, lysosomalenzyme release in leukocytes, and colonic chloride secretion. Musch etal., “Stimulation of Colonic Secretion by Lipoxygenase Metabolites ofArachidonic Acid”, SCIENCE (Washington. D.C.), Vol. 17, pages 1255-1256(1982); Palmer et al., “Chemokinetic Activity of Arachidonic AcidLipoxygenase Products on Leukocytes from Different Species”,PROSTAGLANDINS, Vol. 20, pages 411-448 (1980); Stenson et al.,“Monohydroxyeicosatetraenoic Acids (HETE's) Induce Degranulation ofHuman Neutrophils”, JOURNAL OF IMMUNOLOGY, Vol. 124, pages 2100-2104(1980). These products of arachidonate metabolism are thus potentialmediators of ulcerative colitis and may account for alterations inintestinal fluid and electrolyte secretion.

Recent evidence that the regular intake of n-3 fatty acids from fish oilinhibits neutrophil and monocyte functions suggests that n-3 fatty acidshave antiinflammatory properties. Beneficial effects of marine lipidshave been shown in animal models of inflammatory bowel disease. Empey etal., “Fish Oil-Enriched Diet is Mucosal Protective Against AceticAcid-Induced Colitis in Rats”, CANADIAN JOURNAL OF PHYSIOLOGY ANDPHARMACOLOGY, Vol. 69, pages 480-487 (1991); Vilaseca et al., “DietaryFish Oil Reduces Progression of Chronic Inflammatory Lesions in a RatModel of Granulomatous Colitis”, GUT, Vol. 31, pages 539 (1990). Inpreliminary therapeutic trials, diet supplementation with fish oil hasled to symptomatic improvement of patients with ulcerative colitis, andreduced ethanol-induced damage in human duodenal mucosa. Schepp et al.,“Fish Oil Reduces Ethanol-Induced Damage of the Duodenal Mucosa inHumans”, GASTROENTEROLOGY, Vol. 96, page 446 (1989); Lorenz et al.,“Supplementation with n-3 Fatty Acids from Fish Oil in ChronicInflammatory Bowel Disease”, JOURNAL OF INTERNAL MEDICINE SUPPLEMENT,Vol. 225, pages 225-232 (1989); Hillier et al., “Incorporation of FattyAcids from Fish Oil and Olive Oil into Colonic Mucosal Lipids andEffects Upon Eicosanoid Synthesis in Inflammatory Bowel Disease”, GUT,Vol. 32, pages 1151-1155 (1991); Saloman et al., “Treatment ofUlcerative Colitis with Fish Oil N-3-w-Fatty Acid: An Open Trial”,JOURNAL OF CLINICAL GASTROENTEROLOGY, Vol. 12, No. 2, pages 157-161(1990).

When abundant n-3 fatty acids in the form of fish oil are included inthe diet, eicosapentaenoic (EPA) and docosahexaenoic acid may inhibitthe synthesis of arachidonic acid from linoleic acid, reduce plasmalevels of arachidonic acid by competing for incorporation into membranephospholipids, and compete with arachidonic acid as a substrate forcyclooxygenase, and to a greater extent, lipoxygenase metabolism.Production of the 2-series prostaglandins (PGE₂, TXB₂), and the 4-seriesleukotrienes (LTB₄, LTC₄) are thus diminished, and the less biologicallyactive 3-series prostaglandins (PGE₃, TXB₃) and 5-series leukotrienes(LTB₅, LTC₅) are formed. It is through these mechanisms that dietaryfish oils are thought to manifest their antiinflammatory effects.

Fretland et al., “Eicosanoids and Inflammatory Bowel Disease: Regulationand Prospects for Therapy”, PROSTAGLANDINS LEUKOTRINES AND ESSENTIALFATTY ACIDS, Vol. 41, pages 215-233, at pages 224-225 (1990) relate thatin a small uncontrolled pilot study of ulcerative colitis patients givenfish oil capsules (Max EPA) containing 3-4 grams of EPA daily for twelveweeks showed significant improvement in symptoms and histologicalappearance of the rectal mucosa by the end of the treatment period.Neutrophil LTB₄ levels fell significantly during the treatment. The MaxEPA also contained some vitamin E, a compound with antioxidant andantiinflammatory properties, which could conceivably have accounted forsome of the therapeutic effect. Dietary vitamin E supplementationhowever, was shown not to promote changes in eicosanoid levels obtainedfrom rectal dialysate fluid of active ulcerative colitis patients in aseparate study.

EXPERIMENT 2

A major limitation in investigating the pathogenic mechanismsresponsible for the mucosal injury observed during chronic inflammationof the intestine and colon has been the relative paucity of relevantanimal models. Two models of colitis produced in rats that have receivedmuch attention over the past few years are the acetic acid andtrinitrobenzene sulfonic acid (TNBS) models. The mechanism by whichacetic acid produces the diffuse colitis is thought to involvenonspecific, acid induced injury to the colonic mucosa that is followedby an acute inflammatory response. Apparently the protonated form of theacid is required to induce the colitis since neither HCl (pH 2.3) norsodium acetate (pH 7.0) is effective in eliciting the inflammatoryresponse. However, there is some evidence to suggest that acetic acidmay promote other pathophysiological events (e.g. fluid and electrolytesecretion) using noncytotoxic concentrations of the acid.

Recent studies have demonstrated that the intrarectal administration ofthe hapten, TNBS, in the presence of a mucosal barrier breaker such asethanol, produces an acute and possibly chronic colitis in unsensitizedrats. The mechanism(s) by which buffered or unbuffered TNBS in thepresence of ethanol initiates inflammation in unsensitized animals isunclear; however, it has been suggested to involve macrophage-mediatedrecognition and lysis of TNBS-modified autologous cells within themucosa. However, more recent evidence suggests more complicatedmechanisms. For example, the barrier breaker, ethanol, is an extremelypotent pro-inflammatory solvent alone. Furthermore, it has beendemonstrated that TNBS is metabolized by certain colonic enzymes andsubstrates to yield both pro-inflammatory and cytotoxic oxidants thatcould initiate colonic inflammation. Grisham et al., “Metabolism ofTrinitrobenzene Sulfonic Acid by the Rat Colon Produces Reactive OxygenSpecies”, GASTROENTEROLOGY, Vol. 101, pages 540-547 (1991). A recentstudy directly compared the acetic acid and the TNBS (+ETOH) models ofcolitis and found that either model may be useful to study those eventsthat occur at the time of inflammation (e.g. arachidonate metabolism,granulocyte infiltration and metabolism, etc.) or during repair.However, the use of these models of colitis may have significantlimitations in understanding those immunological events that initiatethe acute and chronic inflammatory episodes. For example, theinflammation and tissue injury observed in human inflammatory boweldisease is most probably a result of inappropriate immunologicalactivation (e.g. autoimmune, infectious agent, etc.) whereas theinflammation induced by the intrarectal application of acetic acid,ethanol or ethanol plus TNBS is a response to extensive mucosal injury.Thus, the mechanisms by which inflammation (and mucosal injury) areachieved in the human disease may be very different than those in theexperimental models.

For these reasons, a model of acute and chronic distal colitis in ratswas developed based upon a previously published method in which purifiedbacterial cell wall polymers (derived from Group A streptococci) areinjected intramurally into the distal colon of genetically-susceptiblerats. Sartor et al., “Granulomatus Entercolitis Induced by PurifiedBacterial Cell Wall Fragments”, GASTROENTEROLOGY, Vol. 89, pages 587-595(1985). This model produces an acute and chronic inflammationcharacterized by the infiltration of large numbers of inflammatorycells, enhanced mucosal permeability, interstitial fibrosis, and mucosalthickening as well as the extraintestinal manifestations of arthritis,hepatic and splenic granulomas. Unlike most models of colitis, theinflammation induced in this model promoting mucosal and submucosalinjury rather than the injury causing the inflammation.

In Experiment 2 the objectives were: (a) to determine whether this modelof colitis responds to sulfasalazine (SAZ) and (b) to assess the effectsof specially formulated enteral diets on the injury and inflammationobserved in the colon, liver and spleen.

Female Lewis rats (150-175 g) were maintained in a controlledtemperature and light-dark cycle (12 hours:12 hours) and housed inwire-mesh bottomed cages and given water and standard laboratory ratchow ad libitum. A total of 48 rats were divided into 6 groups of 8 ratseach consisting of a sham (rats receiving Diet #1 {base diet} with nopeptidoglycan/polysaccharide {PG/PS} injection) control group, a chowgroup and 4 groups of rats placed on 4 different polymeric diets (Table6). Rats received either chow or polymeric diets (320 kcals/kg/day or 60mL of liquid diet/day) for 7 days preceding induction of colitis. Distalcolitis was induced by a modification of the method of Sartor et al inwhich multiple sites (8-9) along the distal colon were injectedintramurally with 60 ul/site to deliver a dose ofpeptidoglycan/polysaccharide (PG/PS) of 12.5 ug/g body weight. Thealbumin (sham) control group received the same number of injections ofhuman serum albumin into the distal colon.

TABLE 6 INFLAMMATORY BOWEL DISEASE FORMULATIONS (Formulations expressedas percentage of calories) Diet^(a) Lipid^(b) Carbohydrate ProteinFiber^(c) Control 18.0% 61.0% 21.0% 14.7% corn oil 42.7% hydrolyzed16.0% caseinates None 2.7% MCT cornstarch 5.0% hydrolyzed 0.6% soy 18.3%sucrose soy^(d) lecithin Fish oil 18.0% 61.0% 21.0% 1.6% canola oil42.7% hydrolyzed 16.0% caseinates None 11.7% fish oil cornstarch 5.0%hydrolyzed 2.7% MCT 18.3% sucrose soy 1.4% soybean oil 0.6% soy lecithinFOS 18.0% 61.0% 21.0% 4.5 g 14.7% corn oil 42.7% hydrolyzed 16.0%caseinates 2.25 g FOS 2.7% MCT cornstarch 5.0% hydrolyzed 2.25 g gum0.6% soy 18.3% sucrose soy arabic lecithin XOS 18.0% 61.0% 21.0% 4.5 g14.7% corn oil 42.7% hydrolyzed 16.0% caseinates 2.25 g XOS 2.7% MCTcornstarch 5.0% hydrolyzed 2.25 g gum 0.6% soy 18.3% sycrose soy arabiclecithin ^(a)The caloric density of all diets is 1.2 kcal/mL. Thenutrient base is 1250 kcal. As used herein “nutrient base” means theamount of calories of a product that must be consumed to provide 100% ofthe US RDA of vitamins and minerals for humans. ^(b)Mochida concentratedfish oil (28% EPA: 12% DHA). ^(c)Fiber is expressed a g/8 fl oz. FOS(Fructooligosaccharide, Golden Technologies Inc.), gum arabic (Nutriloidarabic, TIC Gums), XOS (Xylooligosaccharide, Suntory, Inc.). ^(d)Soyprotein hydrolyzate PP750 (slightly hydrolyzed).

Total dietary intake and body weights of the control and liquid dietgroups were recorded for each 24 hour period during the course of the 4week experiment (1 week prior to the induction of colitis and 3 weeksfollowing PG/PS or albumin injection).

To assess the effects of SAZ in this model, female Lewis rats wereorally administered SAZ immediately following the induction of colitis.Rats were given chow ad libitum for the duration of the four week studyperiod. Similar measurements were assessed as described below.

All rats receiving nutritional therapy or SAZ were euthanized with anoverdose of pentobarbital and the distal colon and cecum were excisedand opened longitudinally. The length and weight of the perfused segmentwere recorded and the tissue divided longitudinally into three stripsfor wet-to-dry ratios, histology and myeloperoxidase (MPO)determinations. Colonic MPO activity was determined in which 3,3′ 5,5′tetramethylbenzidine was used as the electron donating substrate andhexadecyltrimethylammonium hydroxide was used as the detergent. Spleenand liver weights were recorded. Circulating levels of nitrate andnitrite were also quantified using the Griess reagent and used asindices of immune system activation (i.e. activation of thereticuloendothelial system {nitric oxide synthase}). The studies wereanalyzed as a completely randomized design. Treatment differences wereseparated using an F-protected least significant difference (LSD)method. In both studies additional chow fed control animals that weretreated under laboratory conditions described above were incorporated inthe statistical analysis.

Effects of SAZ administration are presented in Table 7. It was foundthat oral administration of SAZ beginning immediately followinginduction of colitis significantly attenuated the increases in MPOactivity and tended to reduce colon weight compared to a chow fed groupreceiving PG/PS suggesting that this antiinflammatory agent inhibitsgranulocyte infiltration and fibrosis (we observed no significantincrease in wet to dry ratios in our inflamed bowel suggesting that theincreases in colon weight were due to collagen deposition). Histologicalinspection of the SAZ-treated tissue confirmed inhibition of leukocyteinfiltration and fibrosis. Previous studies have demonstrated that theinflammation induced by the intramural (subserosal) injection of PG/PSis primarily submucosal in nature and heterogeneous with respect toepithelial cell injury. For example histological inspection of thistissue reveals foci of modest epithelial injury surrounded by regions ofa completely intact epithelium.

In addition to its protective effects on the colon, SAZ treatmentresulted in liver and spleen weights significantly lower than the PG/PStreated animals and comparable to the sham control suggesting that SAZinhibits granuloma development and necrosis in these two organs. Atpresent, the mechanism by which SAZ protects these two organs remainsundefined. It may be that SAZ protects primarily the gut therebyinhibiting the emigration of noxious luminal antigens and bacterialproducts into the portal and systemic circulation where they may promotedistal organic inflammation. Alternatively, SAZ or one of its activemetabolites (e.g. 5-ASA) is present in the systemic circulation and mayexert direct antiinflammatory action on the various organ systems. Theantiinflammatory mechanism of SAZ remains the subject of active debate.There is a large body of experimental data to suggest that the activemoiety of SAZ is 5-ASA which is a potent antioxidant and is a modest5-lipoxygenase inhibitor. In addition, there is emerging evidence tosuggest that the parent diazo compound (ie., SAZ) may possesssignificant antiinflammatory activity.

Recent studies have suggested that nutritional supplementation in theform of enteral diets may prove useful as adjunctive or primary therapyfor patients with IBD. Indeed, recent reports suggest that n-3 fattyacids from fish oil as well as the SCFA produced during the fermentationof indigestible carbohydrates may attenuate some of the pathophysiologyassociated with active gut inflammation. Therefore, we ascertainedwhether three enteral diets, one supplemented with fish oil or twodifferent diets supplemented with two forms of indigestible carbohydratecould inhibit some of the inflammation observed in a model of chroniccolitis. The results are presented in FIGS. 1-5. FIG. 1 presents colonweights of animals following the various therapies (diets). FIG. 2presents MPO activity in colonic tissue of rats following the varioustherapies (diets). FIG. 3 presents liver weights of animals followingthe various therapies (diets). FIG. 4 presents spleen weights in theanimals following the various therapies (diets). FIG. 5 presents levelsin circulating plasma of nitrate and nitrite in animals following thevarious therapies (diets).

TABLE 7 EFFECTS OF SAZ MPO ACTIVITY TREAT- COLON WT (units/cm Liver wtSpleen wt MENT (g dry wt/cm) colon) (mg/g bw) (mg/g bw) Sham Chow .016 ±.003^(b) 1.17 ± 2.5^(b) 31.5 ± 3.1^(b) 2.08 ± 1.1^(b) PG/PS Chow .029 ±.002^(a) 8.49 ± 1.6^(a) 45.0 ± 2.0^(a) 6.95 ± .70^(a) SAZ .021 ±.004^(a,b) 1.40 ± 2.9^(b) 34.0 ± 3.6^(b) 2.92 ± 1.3^(b) ^(c)P = .0045.0195 .0008 .0007 ^(a,b)Least Square means with unlike superscriptletters differ (P < .05). ^(c)= Overall treatment effect.

Results indicate that all enteral diets used in this study provided fora certain degree of antiinflammatory activity. The addition of fish oilor indigestible oligosaccharides such as FOS and XOS demonstratedgreater antiinflammatory activity compared to the base control or chowfed PG/PS rats. Antiinflammatory activity was ascertained using colon,liver and spleen weights along with MPO activity. Results show that thecontrol diets as well as the fish oil and XOS diets produced colonweights that were significantly lower than chow fed PG/PS animals andcomparable to the sham control (FIG. 1). While all enteral diets tendedto attenuate MPO activity compared to chow PG/PS fed animals, only thefish oil and XOS diets were significantly lower than chow animals (FIG.2). Only the chow fed PG/PS rats resulted in an MPO activity that wassignificantly greater than the sham control. Liver weights also wereaffected by the diet (FIG. 3). The FOS and XOS diets resulted in liverweights that were significantly lower than the chow fed PG/PS animals.Only chow fed PG/PS rats had liver weights that were significantlydifferent than sham control animals. The fish oil, FOS and XOS dietsresulted in spleen weights that were comparable to the sham control(FIG. 4). The control and chow diets produced spleen weights that weregreater than the sham control. All enteral diets significantlyattenuated the increase in nitrate and nitrite compared to chow fedanimals. These levels were comparable to sham controls. This is ofimportance because it has been proposed that the large increases incirculating levels of nitrate and nitrite such as in the PG/PS chow fedgroup, arise from the production of nitric oxide by extravasatedpolymorphonuclear cells, monocytes and macrophages.

These results demonstrate that a complete enteral diet (control diet)given to rats for 28 days with distal colitis, reduced disease activityas indicated by the above indices of inflammation. Howeversupplementation with the bioactive ingredients, fish oil, FOS or XOS,showed additional antiinflammatory activity by significantly attenuatingthe colonic and extraintestinal inflammation associated with distalcolitis. In most instances, these indices of inflammation were similarto those indices in chow fed sham controls. The antiinflammatoryactivity of these diets was confirmed via histological inspectionshowing an inhibition of inflammation and maintenance of crypt cellintegrity.

An interesting aspect of the present study is that supplementation ofenteral diets with fish oil or indigestible oligosaccharides rendersthese diets similar in efficacy to a known antiinflammatory drug (SAZ)used to treat human IBD and which has been shown to be efficacious inthis model of inflammation. Although enteral diets may be considered asalternative primary therapy for chronic gut inflammation, it may be moreuseful to consider their use as adjunctive therapy to be used incombination with steroids and/or aminosalicylates.

It is believed to be an important feature of an enteral nutritionalproduct of the present invention that it contains an oil blend whichcomprises, by weight, a total of at least 25% of one or more oilsselected from the group of oils which contain eicosapentaenoic acid(20:5n3) and docosahexaenoic acid (22:6n3). The nutritional product ofthe invention may comprise the oil blend presented in Table 8. Otherfeatures of an oil blend useful in the practice of the present inventionare presented in Table 9 and 10.

TABLE 8 ULCERATIVE COLITIS PRODUCT OIL BLEND (as % of total weight ofoil blend) OIL TARGET PREFERRED RANGE Canola Oil 9.3%  5.0%-40.0% MCT16.2% 10.0%-50.0% Fish Oil 65.0% 25.0%-80.0% Soybean Oil 5.5% 3.0%-30.0% Soy Lecithin 4.0% 2.0%-6.0%

TABLE NO. 9 FATTY ACID PROFILE OF NEW LIPID BLEND (as % of total fattyacids by weight, by analysis) Caproic (6:0) 0.53 Caprylic (8:0) 10.35Capric (10:0) 7.16 Lauric (12:0) 0.29 Myristic (14:0) 3.53 Palmitic(16:0) 7.41 Palmitoleic (16:1n7) 5.73 Stearic (18:0) 1.39 Oleic (18:1n9)15.23 Linoleic (18:2n6) 7.21 Gamma-Linoleic (18:3n6) 0.21 Alpha-linoleic(18:3n3) 2.21 Stearidonic (18:4n3) 2.40 Arachidic (20:0) 0.13 Eicosenoic(20:1n9) 0.74 Arachidonic (20:4n6) 0.87 Eicosapentaenoic (20:5n3) 17.14Erucic (22:1n9) 0.17 Docosapentaenoic (22:5n3) 2.08 Docosahexaenoic(22:6n3) 7.73 Nervonic (24:1n9) 0.14 Others 7.35 TOTAL 100.00

TABLE 10 FATTY ACID LEVELS IN OIL BLEND (percent of total fatty acids)MOST PREFERRED PREFERRED FATTY ACID TARGET RANGE RANGE Oleic acid 13.5%11.5%-15.7% 12.1%-15.1% (18:1n9) Linoleic acid 7.8% 6.6%-9.0% 7.0%-8.6%(18:2n6) Alpha-Linolenic 1.8% 1.5%-2.1% 1.6%-2.0% acid (18:3n3)Eicosapentaenoic 17.8% 15.1%-20.5% 16.0%-19.6% acid (20:5n3)Docosahexaenoic 7.5% 6.3%-8.6% 6.7%-8.3% acid (22:6n3) n-6/n-3 ratio0.32 0.25-4.0  18:2n6/18:3n3 4.26  3.0-10.0 *The n-6 fatty acids whichare used in determining the n-6/n-3 ratio for the product disclosedherein are: Linoleic (18:2n6), Eicosadienoic (20:2n6) and Arachidonic(20:4n-6). The n-3 fatty acids which are used in determining the n-6/n-3ratio for the product disclosed herein are: Alpha-linolenic (18:3n3),Stearidonic (18:4n3), # Eicosapentaenoic (20:5n3), Docosapentaenoic(22:5n3) and Docosahexaenoic (22:6n3).

While not intending to be bound by theory, the combination ofindigestible carbohydrate and specifically dietary fiber andindigestible oligosaccharides with fish oil may increase theincorporation of n-3 fatty acids into colonocytes. The incorporation ofn-3 fatty acids into colonocytes of persons consumingpolymeric/elemental diets devoid of indigestible fermentable material isslow. Hypoproliferation of colonocytes and atrophy is documented withelemental low residue diet feedings. Fermentable indigestibleoligosaccharide such as fructooligosaccharides can promote cellproliferation. Rate and extent of n-3 fatty acid incorporation intocolonocytes is dependent on exchange of plasma n-3 fatty acids and colonmucosal phospholipids and rate of colonic cell turnover. The maintenanceor promotion of cell proliferation due to the incorporation ofindigestible carbohydrate into a liquid diet containing n-3 fatty acidscould promote a rapid increase of n-3 fatty acids from fish oil intocolonic mucosal lipids compared to a liquid diet devoid of indigestiblecarbohydrate. The therapeutic benefit of increasing the incorporation ofn-3 fatty acids into colonic mucosal phospholipids is to (a) promote anantiinflammatory effect by modulating local eicosanoid generation by thegastrointestinal mucosa of ulcerative colitis patients and (b) promotethe rapid incorporation of n-3 fatty acids from fish oil in thegastrointestinal mucosa of ulcerative colitis patients which willdecrease the hyperimmune response resulting in reduced mucosalulceration and disease activity index.

A growing body of data indicates that oxygen derived free radicals suchas superoxide (O₂ ^(.)), hydrogen peroxide (H₂O₂), and hydroxyl radicals(OH^(.)) have a role in mediating intestinal damage in inflammatorybowel disease. The most probable source of these oxidants are thephagocytic leukocytes since these cells are known to be present in largenumbers in the inflamed mucosa and have been shown to producesignificant amounts of reactive oxygen species in response to certaininflammatory stimuli. Grisham, “Role of Neutrophil—Derived Oxidants inthe Pathogenesis of Inflammatory Bowel Disease”, PROGRESS ININFLAMMATORY BOWEL DISEASE, Vol. 12, No. 1, pages 6-8 (1991). Grisham etal., “Neutrophil-Mediated Mucosal Injury. Role of Reactive Metabolites”,DIGESTIVE DISEASES AND SCIENCES, Vol. 33, pages 6-15S (1988), havehypothesized that in ulcerative colitis, transient ischemia-reperfusionepisodes produce high levels of free radicals resulting in mucosalulceration.

Grisham et al., “Oxidant Mechanisms in the Human Colon”, INFLAMMATION,Vol. 14, pages 669-680 (1990) have determined that the normal colon,particularly the mucosa, contains small amounts of antioxidant enzymesystems such as superoxide dismutase, catalase, and GSH peroxidesuggesting that the colon may be susceptible to oxidant-mediated damage.Data, however, on endogenous antioxidant proteins in the intestinalmucosa of patients with inflammatory bowel disease are lacking. The twomost important copper and zinc containing proteins with radicalscavenging potential are metallothionein and superoxide dismutase.Metallothionein is a metal binding protein whose function is theregulation of copper and zinc metabolism. Thornalley et al., “PossibleRole for Metallothionein in Protection Against Radiation—InducedOxidative Stress. Kinetics and Mechanism of its Reaction with Superoxideand Hydroxyl Radicals”, BIOCHIMICA ET BIOPHYSICA ACTA, Vol. 827, pages36-44 (1985), were the first to note the high OH^(.) scavengingpotentials of metallothionein. Since then it has been found to protectDNA molecules, cells in culture, and whole organisms against thedetrimental effects of several types of free radical generatingtreatments. Abel et al., “Inhibition of Hydroxyl-Radical-Generated DNADegradation by Metallothionein”, TOXICOLOGY LETTERS, Vol. 47, pages191-196 (1989); Bakka et al., “Radioresistance in Cells with HighContent of Metallothionein”, EXPERENTIA, Vol. 38, pages 381-383 (1982);Matsubara, “Alteration of Radiosensitivity in Metallothionein InducedMice and a Possible Role of Zn—Cu-Thioneine in GSH—Peroxidase System”,EXPERENTIA, Vol. 52, pages 603-613 (1987). Furthermore, Mulder et al.,“Decrease in Two Intestinal Copper/Zinc Containing Proteins withAntioxidant Function in Inflammatory Bowel Disease”, GUT, Vol. 32, pages1146-1150 (1991), found that superoxide dismutase content was similar incontrol mucosa and non-inflamed mucosa from patients with inflammatorybowel disease but was decreased in inflamed mucosa. Similar results werealso shown with metallothionein. Overall, a decrease in endogenousintestinal protection against oxygen derived radicals in inflammatorybowel disease may contribute to the pathogenesis of the disease.

Considering the compromised antioxidation state of the large bowel ofinflammatory bowel disease patients, it would be beneficial to increaseintakes of vitamins which have antioxidant properties. Vitamins E, C andbeta-carotene are among the most important of these antioxidantvitamins, but the minerals manganese, copper, zinc and selenium are alsorequired for the functional status of the antioxidant enzymes,metallothionein and superoxide dismutase. In a preferred embodiment theenteral nutritional product of the present invention contains at leastone nutrient selected from the group consisting of beta-carotene,vitamin E, vitamin C, taurine and selenium.

An enteral nutritional product according to the present invention hasabout 18.5%-23.5% (most preferably about 21.0%) of total caloriesprovided by protein, about 59.0%-63.0% (most preferably about 61.0%) oftotal calories provided by carbohydrate, and about 16.0%-20.0% (mostpreferably about 18.0%) of total calories provided by fat. Preferablythe protein source contains at least one material selected from thegroup consisting of intact and hydrolyzed (regardless of degree ofhydrolysis) proteins of high biological value. “High biological value”is understood to mean a protein source which provides a full complementof amino acids to the body. An enteral nutritional product according tothe present invention preferably contains about 20 g of indigestiblecarbohydrate per liter. The source of indigestible carbohydrate may beselected from gum arabic, soy polysaccharide, fructooligosaccharides,hydrolyzed inulin, xylooligosaccharides or any other suitable material.

Key features of the nutritional product are presented in Table 11, theamino acid profile of the product is presented in Table 12 and a morecomplete nutrient profile of the enteral nutritional product of thepresent invention is presented in Table 13.

TABLE 11 KEY NUTRIENT FEATURES OF PRODUCT (percentages are % of totalcalories in product from nutrient) NUTRIENT TARGET PREFERRED RANGEProtein 21.0% (67.8 g/L) 18.5%-23.5% (59.7 g/L-75.9 g/L) Carbohydrate 61.0% (201.5 g/L) 59.0%-63.0% (194.9 g/L-208.1 g/L) Fat 18.0% (28.2g/L) 16.0%-18.0% (25.1 g/L-31.3 g/L) Beta-carotene 5,000 ug/L 2,500ug/L-6,500 ug/L Vitamin E 300 IU/L 100 IU/L-450 IU/L Vitamin C 650 mg/L250 mg/L-850 mg/L Taurine 275 mg/L 200 mg/L-350 mg/L Indigestable 19.89g/L 16.91 g/L-22.87 g/L Carbohydrate

TABLE NO. 12 AMINO ACID PROFILE OF PRODUCT (Per actual analysis, valuesnormalized to 100%) AMINO ACID g/100 g Protein Aspartic Acid 7.08Threonine 4.34 Serine 5.68 Glutamic Acid 20.58 Proline 10.55 Glycine1.81 Alanine 3.04 Valine 5.90 Methionine 2.78 Isoleucine 4.77 Leucine9.08 Tyrosine 4.79 Phenylalanine 4.96 Histidine 2.67 Lysine 7.27Arginine 3.15 Tryptophan 0.99 Cystine 0.56

TABLE 13 PREFERRED NUTRIENT PROFILE OF THE ULCERATIVE COLITISNUTRITIONAL PRODUCT TARGET ACCEPTABLE RANGE/QUANTITY QUANTITY/ QUANTITY/NUTRIENT LITER LITER Protein, g 67.4 66-70 Fat, g 27.2 25-29Carbohydrate, g 207 204-215 Total Dietary 10.7  9.1-12.3 Fiber, gIndigestible 12.4 10.5-14.3 Oligosacharide (FOS), g Gum Arabic, g 9.1 7.7-10.5 Soy Polysaccharide, g 1.6 1.4-1.8 β-carotene, μg 50002500-6500 Vitamin A, IU 5500 4500-6500 Vitmain D, IU 800 675-950*Vitamin E, IU 300 100-450 Vitamin K₁, μg 135 120-150 Vitamin C, mg 650250-850 Folic Acid, μg 1900 1688-2150 Thiamine, mg 6.5 2.53-8.0 Riboflavin, mg 5 2.87-6.5  Vitamin B₆, mg 5 3.38-6.5  Vitamin B₁₂, μg 1810.1-25.0 Niacin, mg 40 33.8-50.0 Choline, mg 525 506-900 Biotin, μg 750 506-1000 Pantothenic Acid, mg 24 16.9-30   Sodium, mg 1500 1350-1650Potassium, mg 2000 1800-2200 Chloride, mg 1519 1367-1671 Calcium, mg1800 1477-1920 Phosphorus, mg 1250 1055-1372 Magnesium, mg 450 422-550Iodine, μg 175 158-300 Copper, mg 2.61 2.25-3.0  Zinc, mg 29.2 25.3-35.0Iron, mg 22.2 20.3-25.0 Selenium, μg 90 78.8-125  Chromium, μg 125112.5-150   Molybendum, μg 206 168.8-250   Carnitine, mg 150 127-200Taurine, mg 275 200-350 Kcal/mL 1.29 1.27-1.34 *d-alpha-tocopheryl (allnatural form) or dl-alpha tocopehrol acetate, or a combination of thetwo

The Bill of Materials for manufacturing an enteral nutritional productin accordance with the present invention is presented in Table 14. It isunderstood that various changes in ingredients and quantities may bemade without departing from the scope of the invention.

TABLE 14 BILL OF MATERIALS BATCH SIZE = 45,360 Kgs (100,000 LBS)INGREDIENT AMOUNT WATER 31,605.21 Kgs GUM ARABIC 437.84 KgsULTRATRACE/TRACE MINERAL PREMIX 14.50 Kgs ZINC SULFATE 2969.89 gmsFERROUS SULFATE 2856.50 gms MAGANESE SULFATE 784.60 gms CUPRIC SULFATE423.11 gms SODIUM MOLYBDATE 21.39 gms CHROMIUM CHLORIDE 20.80 gms SODIUMSELENITE 8.11 gms CITRIC ACID 894.94 gms SUCROSE (Carrier) 6520.67 gmsPOTASSIUM CITRATE 50.00 Kgs SODIUM CITRATE 95.00 Kgs POTASSIUM IODIDE9.00 gms POTASSIUM CHLORIDE 91.00 Kgs CORN SYRUP SOLIDS 5630.96 KgsMALTODEXTRIN 1407.52 Kgs MAGNESIUM PHOSPHATE DIBASIC 131.00 Kgs CALCIUMPHOSPHATE TRIBASIC 47.50 Kgs (PREFERABLY MICRONIZED) CALCIUM CARBONATE122.50 Kgs SUGAR (SUCROSE) 852.77 Kgs FRUCTOOLIGOSACCHARIDE 509.96 KgsMEDIUM CHAIN TRIGLYCERIDES 172.69 Kgs (FRACTIONED COCONUT OIL) CANOLAOIL 99.13 Kgs SOY OIL 58.63 Kgs 57% VITAMIN A PALMITATE 250.00 gms 2.5%VITAMIN D 35.00 gms D-ALPHA-TOCOPHERYL ACETATE (R, R, R) 10.65 KgsPHYLLOQUINONE 6.50 gms 30% BETA-CAROTENE 824.00 gms SOY LECITHIN 42.64Kgs SODIUM CASEINATE 1427.04 Kgs PARTIALLY HYDROLYZED SODIUM 1427.04 KgsCASEINATE SOY POLYSACCHARIDE 85.28 Kgs 75% WHEY PROTEIN CONCENTRATE184.46 Kgs REFINED DEODORIZED SARDINE OIL 692.87 Kgs ASCORBIC ACID 37.08Kgs 45% POTASSIUM HYDROXIDE 25.96 Kgs TAURINE 12.00 Kgs WATER SOLUBLEVITAMIN PREMIX 4.50 Kgs NIACINAMIDE 1688.60 gms CALCIUM PANTOTHENATE1092.24 gms THIAMINE CHLORIDE HYDROCHLORIDE 278.78 gms PYRIDOXINEHYDROCHLORIDE 268.34 gms RIBOFLAVIN 217.87 gms FOLIC ACID 37.82 gmsBIOTIN 32.87 gms CYANOCOBALAMIN 0.75 gms DEXTROSE (Carrier) 882.74 gmsFOLIC ACID 43.50 gms CHOLINE CHLORIDE 25.00 Kgs L-CARNITINE 7.00 KgsARTIFICIAL STRAWBERRY FLAVOR 31.75 Kgs ARTIFICIAL CREAM FLAVOR 18.14 KgsFD & C Red Dye No. 3 1,220.16 gms

The liquid nutritional product of the present invention has beenmanufactured by preparing three slurries which are blended together,combined with refined deodorized sardine oil, heat treated,standardized, packaged and sterilized. The process for manufacturing45,360 Kgs (100,000 pounds) of the liquid nutritional product, using theBill of Materials from Table 11, is described in detail below.

A carbohydrate/mineral slurry is prepared by first heating about 6,260Kgs of water to a temperature in the range of about 71 to 77° C. withagitation. The gum arabic is then added to the water using a mixingapparatus. Next the ultratrace/trace mineral premix is added to thewater and dissolved by agitating the resultant solution for at least oneminute. The following minerals are then added, in the order listed, withhigh agitation: Potassium Citrate, Sodium Citrate, Potassium Iodide andPotassium Chloride. The corn syrup solids and maltodextrin are thenadded to the slurry and the temperature of the slurry is maintained atabout 71° C. with high agitation for at least about 20 minutes. Theproduct has been manufactured using maltodextrin distributed by GrainProcessing Corporation, Muscatine, Iowa, U.S.A. under the tradedesignation “Maltrin M-100” and corn syrup solids distributed by GrainProcessing Corporation under the trade designation “Maltrin M-200”. Addthe Magnesium Phosphate Dibasic, Calcium Phosphate Tribasic, and CalciumCarbonate to the slurry. The sugar (sucrose), and Fructooligosaccharideare added to the slurry. The product has been manufactured usingfructooligosaccharide powder distributed by Golden Technologies Company,Golden, Colo., U.S.A. under the trade designation “Nutriflora-PFructo-oligosaccharide Powder (96%)”. The completed carbohydrate/mineralslurry is held with high agitation at a temperature in the range ofabout 60 to 66° C. for not longer than 12 hours until it is blended withthe other slurries.

An oil slurry is prepared by combining and heating the medium chaintriglycerides (fractionated coconut oil), canola oil and soy oil to atemperature in the range of about 32 to 43° C. with agitation. The 57%Vitamin A Palmitate, 2.5% Vitamin D₃, D-alpha-tocopheryl acetate (R,R,Rform), phylloquinone and 30% beta-carotene are added to the slurry withagitation. The product has been manufactured using D-alpha tocopherylAcetate distributed by Distillation Products Industries, a division ofEastman Kodak Chemical Company, Rochester, N.Y. U.S.A. under the tradedesignation “Eastman Vitamin E 6-81 D-Alpha Tocopheryl AcetateConcentrate”. The soy lecithin is then added to the slurry withagitation. The completed oil slurry is held under moderate agitation ata temperature in the range of about 32 to 43° C. for not longer than 12hours until it is blended with the other slurries.

A protein-and-fiber-in-water slurry is prepared by first heating about19,678 Kgs of water to a temperature in the range of about 60 to 63° C.with agitation. The sodium caseinate, partially hydrolyzed sodiumcaseinate and soy polysaccharide are blended into the slurry using amixing apparatus. The product has been manufactured using a partiallyhydrolyzed sodium caseinate distributed by New Zealand Milk Products,Santa Rosa, Calif., U.S.A. under the trade name Alanate 167. Thetemperature of the slurry is lowered to about 57 to 60° C. and then the75% whey protein concentrate is added to the slurry using a mixingapparatus. The completed protein-and-fiber-in-water slurry is held underagitation at a temperature in the range of about 54 to 60° C. for notlonger than 2 hours before being blended with the other slurries.

The oil slurry and the protein-and-fiber-in-water slurry are blendedtogether with agitation and the resultant blended slurry is maintainedat a temperature in the range of about 54 to 66° C. After waiting for atleast one minute the carbohydrate/mineral slurry is added to the blendedslurry from the preceding step with agitation and the resultant blendedslurry is maintained at a temperature in the range of about 54 to 66° C.The vessel which contained the carbohydrate/mineral slurry should berinsed with about 220 Kgs of water and the rinse water should be addedto the blended slurry. The refined deodorized sardine oil is then addedto the slurry with agitation. (It is believed that in a most preferredmethod of manufacture the sardine oil would be slowly metered into theproduct as the blend passes through a conduit at a constant rate.) Theproduct has been manufactured using deodorized sardine oil distributedby Mochida International Company, Limited, Shinjuku-ku, Tokyo, Japanunder the trade designation “50% Omega-3 marine oil EPA:DHA 28:12 with0.8% mixed tocopherol as antioxidant”. Preferably after at least 5minutes the pH of the blended slurry is determined. If the pH of theblended slurry is below 6.55, it is adjusted with dilute potassiumhydroxide to a pH of 6.55 to 6.8.

After waiting a period of not less than one minute nor greater than twohours the blended slurry is subjected to deaeration,Ultra-High-Temperature (UHT) treatment, and homogenization, as describedbelow:

A. Use a positive pump for supplying the blended slurry for thisprocedure.

B. Heat the blended slurry to a temperature in the range of about 66-71°C.

C. Deaerate the blended slurry to 25.4-38.1 cm of Hg.

D. Emulsify the blended slurry at 61-75 Atmospheres.

E. Heat the blended slurry to a temperature in the range of about 120 to122° C. by passing it through a plate/coil heat exchanger with a holdtime of approximately 10 seconds.

F. UHT heat the blended slurry to a temperature in the range of about144 to 147° C. with a hold time of approximately 5 seconds.

G. Reduce the temperature of the blended slurry to be in the range ofabout 120-122° C. by passing it through a flash cooler.

H. Reduce the temperature of the blended slurry to be in the range ofabout 71 to 82° C. by passing it through a plate/coil heat exchanger.

I. Homogenize the blended slurry at about 265 to 266 Atmospheres.

J. Pass the blended slurry through a hold tube for at least 16 secondsat a temperature in the range of about 74 to 85° C.

K. Cool the blended slurry to a temperature in the range of about 1 to7° C. by passing it through a large heat exchanger.

Store the blended slurry at a temperature in the range of about 1 to 7°C., preferably with agitation.

Preferably at this time appropriate analytical testing for qualitycontrol is conducted. Based on the test results an appropriate amount ofdilution water (10-38° C.) is added to the blended slurry withagitation.

A vitamin solution, a flavor and a color solution are preparedseparately and then added to the blended slurry.

The vitamin solution is prepared by heating about 394 Kgs of water to atemperature in the range of about 43 to 66° C. with agitation, andthereafter adding the following ingredients, in the order listed:Ascorbic Acid, 45% Potassium Hydroxide, Taurine, Water Soluble VitaminPremix, Folic Acid, Choline Chloride, and L-Carnitine. The vitaminsolution is then added to the blended slurry with agitation.

The flavor solution is prepared by adding the artificial strawberryflavor and artificial cream flavor to about 794 Kgs of water withagitation. A nutritional product according to the present invention hasbeen manufactured using an artificial strawberry flavor distributed byFirmenich Inc., Princeton, N.J. U.S.A. under the trade designation “Art.strawberry 57.883/A” and an artificial cream flavor distributed byFirmenich Inc. under the trade designation “Art Cream 59.200/A”. Theflavor solution is then added to the blended slurry with agitation.

A color solution is prepared by adding the FD&C Red Dye No. 3 to about121 Kg of water with agitation. The color solution is then added to theblended slurry with agitation.

If necessary, diluted potassium hydroxide is added to the blended slurrysuch that the product will have a pH in the range of 6.4 to 7.0 aftersterilization. The completed product is then placed in suitablecontainers and subjected to sterilization. Of course, if desired asepticprocessing could be employed.

A method of improving the nutritional status and reversing thecharacteristic diarrhea and inflammatory condition in a mammaliancreature, such as a human, having ulcerative colitis or inflammation ofthe colon comprises enterally feeding to such a mammalian creature, orhuman, a therapeutically effective amount of the nutritional productdisclosed herein.

Clinical trials evaluating the enteral nutritional product disclosedherein in humans will begin in the near future and data supporting thebeneficial properties of the instant invention will be provided. It isexpected that this data will confirm the positive effect of the productdisclosed herein upon ulcerative colitis.

While certain representative embodiments have been described herein forthe purpose of illustrating the invention, it is understood that personsof skill in the art can make various modifications to these illustrativeembodiments without deviating from the scope of the invention.

What is claimed is:
 1. A nutritional product for enteral feedingcomprising in combination: (a) a source of indigestible carbohydratewhich is metabolized to short chain fatty acids by microorganismspresent in the human colon and which comprises at least one materialselected from the group consisting of dietary fibers and indigestibleoligosaccharides; and (b) an oil blend containing certain fatty acids,expressed as percentages by weight of total fatty acids in the oilblend, as follows: FATTY ACID % OF TOTAL FATTY ACIDS Oleic acid (18:1n9)11.5-15.7 Linoleic acid (18:2n6) 6.6-9.0 Alpha-Linolenic acid (18:3n3)1.5-2.1 Eicosapentaenoic acid (20:5n3) 15.1-20.5 Docosahexaenoic acid(22:6n3)  6.3-8.6.


2. A nutritional product according to claim 1 comprising an oil blendcontaining certain fatty acids, expressed as percentages by weight oftotal fatty acids in the oil blend, as follows: FATTY ACID % OF TOTALFATTY ACIDS Oleic acid (18:1n9) 12.1-15.1 Linoleic acid (18:2n6) 7.0-8.6Alpha-Linolenic acid (18:3n3) 1.6-2.0 Eicosapentaenoic acid (20:5n3)16.0-19.6 Docosahexaenoic acid (22:6n3) 6.7-8.3


3. A nutritional product according to claim 1 comprising an oil blendcontaining certain fatty acids, expressed as percentages by weight oftotal fatty acids in the oil blend, as follows: % OF TOTAL FATTY FATTYACID ACIDS Oleic acid (18:1n9) About 15.2 Linoleic acid (18:2n6) About7.2 Alpha-Linolenic acid (18:3n3) About 2.2 Eicosapentaenoic acid(20:5n3) About 17.1 Docosahexaenoic acid (22:6n3) About 7.7


4. A nutritional product according to any of claim 1, 2 or 3 wherein, byweight, the ratio of the sum of all of the n-6 fatty acids in the oilblend to the sum of all of the n-3 fatty acids in the oil blend is inthe range of 0.25 to 4.0.
 5. A nutritional product according to any oneof claim 1, 2 or 3 wherein, by weight, the ratio of Linoleic acid(18:2n6) in the oil blend to Alpha-Linolenic acid (18:3n-3) in the oilblend is in the range of 3.0-10.0.
 6. A nutritional product according toclaim 4 wherein, by weight, the ratio of Linoleic acid (18:2n6) in theoil blend to Alpha-Linolenic acid (18:3n-3) in the oil blend is in therange of 3.0-10.0.
 7. A nutritional product according to claim 1 whereinthe source of indigestible carbohydrates comprises at least one materialselected from the group consisting of gum arabic, soy polysaccharide,fructooligosaccharides, hydrolyzed inulin and xylooligosaccharides.
 8. Anutritional product according to claim 1 further comprising at least onenutrient selected from the group consisting of beta-carotene, vitamin E,vitamin C, taurine and selenium.
 9. A nutritional product according toclaim 7 further comprising at least one nutrient selected from the groupconsisting of beta-carotene, vitamin E, vitamin C, taurine and selenium.10. A nutritional product for enteral feeding comprising in combination:(a) an oil blend which comprises, by weight, a total of at least 25% ofone or more oils which contain: Eicosapentaenoic acid (20:5n3) andDocosahexaenoic acid (22:6n3); (b) a source of indigestible carbohydratewhich is metabolized to short chain fatty acids by microorganismspresent in the human colon and which comprises at least one materialselected from the group consisting of dietary fibers and indigestibleoligosaccharides; and (c) at least on nutrient selected form the groupconsisting of beta-carotene, vitamin E, vitamin C, taurine and selenium.11. A nutritional product according to claim 10 in which the oils whichcontain Eicosapentaenoic acid (20:5n3) and Docosahexaenoic acid (22:6n3)are selected from the group consisting of fish oils, marine oils, algaeoils, fungal oils, and vegetable oil.
 12. A nutritional productaccording to claim 10 wherein the source of indigestible carbohydratesis a combination of at least one dietary fiber and at least oneindigestible oligosaccharide.
 13. A nutritional product according toclaim 10 wherein the source of indigestible carbohydrate contains atleast one material selected from the group consisting of gum arabic, soypolysaccharide, fructooligosaccharides, hydrolyzed inulin andxylooligosaccharides.
 14. A nutritional product for enteral feedingcomprising in combination: (a) an oil blend comprising by weight about5-40% canola oil, about 10-50% medium chain triglycerides, about 25-80%fish oil, about 3-30% soybean oil, and about 2-6% soy lecithin; (b) asource of indigestible carbohydrate which is metabolized to short chainfatty acids by microorganisms present in the human colon and whichcomprises at least one material selected from the group consisting ofgum arabic, soy polysaccharide, fructooligosaccharides andxylooligosaccharides; (c) at least one nutrient selected from the groupconsisting of beta-carotene, vitamin E, vitamin C, taurine and selenium;and (d) a source of protein.
 15. A nutritional product according toclaim 14 wherein the oil blend further comprises at least one oilselected from the group consisting of corn oil, safflower oil,high-oleic safflower oil, high-oleic sunflower oil, olive oil, borageoil, black currant seed oil and evening primrose oil.
 16. A nutritionalproduct according to claim 14 wherein, by weight, the ratio of the sumof all of the n-6 fatty acids in the oil blend to the sum of all of then-3 fatty acids in the oil blend is in the range of 0.25 to 4.0.
 17. Anutritional product according to any one of claim 14, 15 or 16 wherein,by weight, the ratio of Linoleic acid (18:2n6) in the oil blend toAlpha-Linolenic acid (18:3n-3) in the oil blend is in the range of 3.0to 10.0.
 18. An enteral nutritional product according to claim 14 havingabout 18.5%-23.5% of total calories provided by protein, about59.0%-63.0% of total calories provided by carbohydrate, and about16.0%-20.0% of total calories provided by fat.
 19. An enteralnutritional product according to claim 14 wherein the source of proteincomprises at least one material selected from the group consisting ofintact and hydrolyzed proteins of high biological value.
 20. Thenutritional product according to claim 1 further comprising a source ofprotein and a source of digestible carbohydrate.
 21. The nutritionalproduct according to claim 20 in which said indigestible carbohydrate ispresent in the quantity of about 16.9 to about 22.8 grams per liter. 22.The nutritional product according to claim 21 in which said proteinprovides about 18.5% to about 23.5% of total calories, saidcarbohydrates provide about 59% to about 63% of total calories, and saidfat provides about 16 to about 18% of total calories.
 23. A method forproviding nutrition to a patient with ulcerative colitis comprising theadministration of a nutritional product according to claim 1 to saidpatient.
 24. The nutritional product according to claim 10 furthercomprising a source of protein and a source of digestible carbohydrate.25. The nutritional product according to claim 24 in which said proteinprovides from about 18.5% to about 23.5% of total calories, saidcarbohydrates provide about 59% to about 63% of total calories, and saidfat provides about 16 to about 18% of total calories.
 26. Thenutritional product according to claim 25 in which said indigestiblecarbohydrate is present in the quantity of about 16.9 to about 22.8grams per liter.
 27. A method for providing nutrition to a patient withulcerative colitis comprising the administration of a nutritionalproduct according to claim 10 to said patient.
 28. A method forproviding nutrition to a patient with ulcerative colitis comprising theadministration of a nutritional product according to claim 26 to saidpatient.
 29. The nutritional product according to claim 14 furthercomprising a source of protein and a source of digestible carbohydrate.30. The nutritional product according to claim 29 in which saidindigestible carbohydrate is present in the quantity of about 16.9 toabout 22.8 grams per liter.
 31. The nutritional product according toclaim 30 in which said protein provides from about 18.5% to about 23.5%of total calories, said carbohydrates provide from about 59% to about63% of total calories, and said fat provides from about 16 to about 18%of total calories.
 32. A method for providing nutrition to a patientwith ulcerative colitis comprising the administration of a nutritionalproduct according to claim 14 to said patient.
 33. A method forproviding nutrition to a patient with ulcerative colitis comprising theadministration of a nutritional product according to claim 31 to saidpatient.
 34. The nutritional product according to claim 8 in which saidbeta-carotene is present in the quantity of about 2,500 to about 6,500ug/liter.
 35. The nutritional product according to claim 8 in which saidvitamin E is present in the quantity of about 100 to about 450 iu/liter.36. The nutritional product according to claim 8 in which said vitamin Cis present in the quantity of about 250 to about 850 mg/liter.
 37. Thenutritional product according to claim 8 in which said taurine ispresent in the quantity of about 200 to about 350 mg/liter.
 38. Thenutritional product according to claim 14 in which said beta-carotene ispresent in the quantity of about 2,500 to about 6,500 ug/liter.
 39. Thenutritional product according to claim 14 in which said vitamin E ispresent in the quantity of about 100 to about 450 iu/liter.
 40. Thenutritional product according to claim 14 in which vitamin C is presentin the quantity of about 250 to about 850 mg/liter.
 41. The nutritionalproduct according to claim 14 in which said taurine is present in thequantity of about 200 to about 350 mg/liter.